Herpes viruses comprise a large family of complex, double-stranded DNA viruses, a number of which are serious human pathogens. Herpes DNA replication requires a group of virally encoded proteins, and is the target of several antiviral drugs. However, the mechanism of Herpes replication is remarkably complex and not well understood. The broad, long-term goal of these studies is to obtain a detailed biochemical understanding of the mechanisms of Herpes primase and polymerase, and how they function together to initiate the synthesis of new DNA strands. This knowledge will, in turn, substantially enhance our understanding of the mechanism by which Herpes viruses replicate their DNA. The specific aims of this proposal are: 1. Obtain a detailed understanding of the role of UL8 during primer synthesis. Even though this protein is essential for Herpes replication, little is known about how it functions mechanistically. 2. Develop a detailed understanding of how primase decides whether or not to polymerize a NTP. Primase is an astoundingly inaccurate enzyme, yet it is very capable of discriminating against NTPs containing modified bases. These studies will elucidate the fidelity mechanisms that give rise to these unusual properties 3. Determine how primase interacts with the sugar of a nucleotide and then synthesize potent and specific inhibitors of this enzyme. Insights obtained from the studies on primase fidelity (Aim 2) will be combined with the results of studies on the interaction of primase with sugar analogs to develop novel inhibitors. 4. Elucidate the mechanism of primase-coupled DNA polymerase activity. These studies will determine how primase and polymerase function together to initiate the synthesis of new DNA strands. 5. Determine how Herpes polymerase discriminates between correct and incorrect dNTPs. Fidelity of DNA replication is one of the most fundamental issues facing DNA polymerases. These studies will determine how Herpes polymerase obtains fidelity during DNA replication. To accomplish these aims, steady state and pre-steady state kinetic approaches, photoactivateable crosslinking reagents, and DNA footprinting methodologies will be used. Additionally, a large number of novel nucleotides will be synthesized.